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The targeted development of a new generation Vaccine for Schistosomiasis

Final Report Summary - THESCHISTOVAC (The targeted development of a new generation Vaccine for Schistosomiasis)

Executive Summary:
SUMMARY
Schistosomiasis is an exceptionally chronic infection caused by the trematode worms of Schistosoma mansoni, S. haematobium and S. japonicum. More than 200 million people in many of the most resource‐deprived regions of the developing world are chronically infected with blood‐dwelling schistosome worms. Infection with these worms can cause chronic debilitating morbidity and result in a massive economic and social burden that is often underestimated, and treatment does not prevent reinfection, indicating a real need for a vaccine. The objectives of TheSchistoVac, carried out by a unique consortium of European and African participants, are to identify molecules on the skin stage schistosomula that can induce protective immune responses. Candidates selected from post-genomic analysis which are recognized by antibodies in human sera collected in endemic African areas were produced as recombinant proteins. These candidates were tested in in vitro parasite opsonisation assays and an in vivo models of vaccination in rats. Uniquely, the reactivity of candidate antigens at the cellular level with human T and B cells was as assessed using samples from newly collected endemic cohorts to discover protective cellular responses. The combined data point out a number of novel vaccine candidates that may be developed into an effective prophylactic vaccine against schistosome infections. By intense training and exchange programs between the participants of the project TheSchistoVac supported the formation of a nucleus of African scientists in helminth vaccine development.

The main findings, achievements and impact of TheSchistoVac are:
• Selection of promising candidates by antibody screening of defined resistant and susceptible populations. Both protein and glycan targets giving robust IgG responses have been identified by HT antibody screening. All are of potential interest for further vaccine development research.
• Definition of responses to candidate antigens by T cells of endemic population classified as resistant and susceptible. Our data suggests that a higher ratio of Th1/Th2 or pro/anti-inflammatory cytokines in response to specific candidate antigens correlates with resistant phenotype. The most promising candidates on basis to the cellular work are SmSP7 and SmLy6b/CD59b.
• Pre‐clinical in vivo testing in the rat model indicates that SmKK7 and a specific glycan type are the most promising vaccine candidates. These candidates will be useful starting points for further pre-clinical and ultimately clinical vaccine development.
• Clearly, different candidates for a vaccine against Schistosoma mansoni infection were selected on the basis of different in vitro and in vivo tests designed to correlate different aspects of the immune response with protective potential. These candidates should be further developed pre-clinically, ideally by comparison with benchmark candidates such SmTSP-2 in the same tests.
• Regulatory T-cell numbers in infected subjects decrease upon chemotherapy, indicating that current infections prevent efficient T-cell response to vaccination.
• Schistosome infection is associated with regulatory B cells and with memory B cells that in particular contribute to IgG4, both of these B cells can contribute prolonging parasite survival.
• A template for development of vaccines against S. haematobium and S. japonicum. The established pipeline for S. mansoni, and the identification of homologous proteins in other schistosome species has provided a development platform.
• TheSchistoVac results led to the advancement of immunological understanding of schistosomiasis (innate and adaptive immunity).
• TheSchistoVac led to improvements in rational vaccine design in schistosomiasis and other helminth infections.
• TheSchistoVac has significantly contributed to sustained and integrated helminth research in Sub‐Saharan Africa.


Project Context and Objectives:
CONTEXT
The overall goal of TheSchistoVac, is to develop novel safe and effective candidates for a second generation vaccine for schistosomiasis. The project aims at the development of candidates for prophylactic vaccines for this major helminth infection by utilising state-of-the-art technologies including genomics, glycomics and bioinformatics, essential involvement of four different disease endemic countries, pre-clinical in vitro and in vivo testing, and SME participation.

Context:
Schistosomiasis is an exceptionally chronic infection caused by the trematode worms of Schistosoma mansoni, S. haematobium and S. japonicum. More than 200 million people in many of the most resource‐deprived regions of the developing world are chronically infected with blood‐dwelling schistosome worms. Infection with these worms can cause chronic debilitating morbidity and result in a massive economic and social burden that is often underestimated. The current strategy for control of infection and morbidity of schistosomiasis depends on repeated mass treatments with the only available drug, praziquantel, a safe single dose drug treatment that is effective against all species of schistosomes. A major impediment to the sustainable success of this strategy is that rapid reinfection occurs after treatment, particularly in young children living in endemic areas. Thus, adequate schistosomiasis control would require, if it were possible, the repeated administration of hundreds of millions of drug treatments on a regular basis. Moreover, continuous mass treatments eventually will induce praziquantel‐resistant strains of schistosomes and there is evidence of increased rebound morbidity. Although new drugs would be useful in case resistance develops, even new drugs cannot prevent rapid re‐infection after treatment, indicating a real need for a vaccine.
Schistosomes ensure their long term survival by a sophisticated, almost perfect, array of immune evasion mechanisms. However, despite their strategies for survival, careful human studies in disease endemic areas over the last 30 years have shown that an important form of natural human immunity does occur against all three schistosome species that cause major clinical disease, vouching for the feasibility of vaccine development. Important data obtained prior to this project indicate that human immunity may be directed against the early post‐penetration skin stage larvae. Although antigens expressed by schistosome larvae have been studied before as vaccine candidates, no success has been booked with respect to the identification of single antigens that are effective in vaccination. It has become clear that the pattern of expression of antigens on life cycle stages might be a very important consideration that could affect the level of immune reactivity to a vaccine candidate. This knowledge has been incorporated into the strategy of TheSchistoVac that aims to identify such a prophylactic vaccine.

Based on previous observations, we propose that two mechanisms are involved in the induction of immune responses against the early larval stage (skin-stage) and these should be considered in the development of an efficient vaccine:
1 Adult worms occasionally release antigens into the host environment, particularly upon dying. The longevity of adult worms means its takes years for infected individuals to gain repeated experience of dying adult worms. Some of these worm antigens are cross-reactive with, or identical to, exposed antigens of the skin-stage schistosomula and skin stage-adult worm cross-reactive or shared antigens are therefore important targets for vaccine development.
2 While human immunity may be directed against the early post-penetration larvae, such protective responses induced are often suppressed by specific cross-reactivity with antigens released from eggs. This will not be the case for unique skin-stage antigens that do not cross-react with egg antigens. Therefore uniquely skin-stage specific antigens also form important targets for vaccine development.
To identify the unique skin-stage molecules, as well as adult worm cross-reactive and shared skin-stage molecules that should form candidates for an effective prophylactic vaccine against schistosome infections TheSchistoVac follows a stepwise procedure of:
(A) initial post-genomic selection of targets
(B) subsequent selection by antibody screening of defined resistant and susceptible populations
(C) pre-clinical in vitro evaluation in schistosomula killing assays, and at the human cellular level
(D) pre-clinical in vivo testing in animal models

The availability of schistosome genome data and the technical ability to screen, select, and evaluate molecules from the whole schistosome transcriptome, has provided the research community with appropriate innovative tools needed for human anti-schistosomiasis vaccine. Previously participants in the consortium have generated a microarray with 37,632 elements containing 35,437 S. mansoni 50-mer oligonucleotide sequences. This microarray has been used to determine the transcription profiles of each S. mansoni gene over 15 life stages including skin-stage schistosomula, worms and eggs. Mining of this unique data set through advanced bioinformatics provides us with the opportunities to pre-select antigens that will allow the rapid screening of biologically and immunologically plausible candidates, cutting down on years of work that would normally have to be undertaken through a more traditional approach of vaccine development.
Usually, vaccine target discovery approaches only take into account proteins as potential immunogens. In schistosomiasis as well as many other infectious diseases glycans (carbohydrates) expressed by the pathogen as part of a polysaccharide (bacteria) or glycoprotein (helminths, protozoa), give rise to strong antibody responses. Protection-correlated responses have been reported for so far unidentified glycan antigens of schistosomes and it has been shown that passive protection could be transferred with an anti-glycan monoclonal. We therefore propose to include schistosomal glycans when searching for the best vaccine candidate.

We primarily use the rat model for immunisation and protection studies with candidate antigens, because humans and rats share critical immunological effector mechanisms against S. mansoni. Infection and re-infection studies in rats, which are semi-permissive hosts, revealed specific antibodies to be the major players in the protective activity against S. mansoni, with the larval stages being sensitive to the action of these antibodies. Remarkably, analogous findings were observed in humans that were immune against re-infection. These findings were supported by in vitro studies showing that human antibodies of the IgG1, IgG3 and IgE isotype as well as rat antibodies (IgG2a and IgE) are cytotoxic for skin-stage schistosomula in the presence of effector cells, including eosinophils, macrophages and platelets. This similarity outlines the usefulness of the rat model of experimental schistosomiasis with regard to our understanding of humoral responses. In the context of TheSchistoVac, we will therefore primarily explore the rat model in order to evaluate the capacity of candidate molecules to protect from S. mansoni infection.

At the in vitro level, rat, human and mouse antibodies and immune cells have been used to test their capability in the ADCC of schistosomula as mentioned above. The in vitro approach provides an ideal tool to measure an important correlate of protection: killing of skin-stage schistosomula mediated by antisera to candidate antigens. We will use schistosomula to assess the opsonising effect of schistosome-specific antibodies followed by attachment of added inflammatory cells via Fc receptors to the parasites (in vitro opsonisation assay).
Finally, the screening of the candidate antigens in endemic populations for immunological recognition at the T and B cell level will utilise the state of the art methodologies to ensure the generation of a maximum level of information on the candidate vaccines, and therefore maximize successful implementation.
A robust CD4+ memory cell response is important for eliciting a strong and long lasting antibody response to a pathogen either as a result of natural immunity or vaccination. The intracellular cytokine analysis in these cells will allow us to determine the type of effector and memory responses that may be elicited to candidate antigens. By measuring effector and memory CD4+ subsets and their intracellular cytokine production in response to vaccine candidates in resistant and susceptible populations, it will be possible to further select the candidates that are capable of eliciting a strong memory T cell response with little Th2 cytokines. Perhaps a characteristic adult (resistance correlated) response not shared with children (susceptibility correlated) can be identified. Immunological memory is an important aspect of not only T but also B cells responses, particularly when considering vaccination. The combined T- and B-cell information is needed for the assessment of how vaccine candidates rank in potential introduction into clinical trials.

Main objectives:
The objectives of TheSchistoVac project are to identify unique skin stage molecules, as well as adult worm cross‐reactive skin stage molecules, which are recognized by antibodies of defined resistant populations. These candidates will be tested in in vitro parasite killing assays and in vivo models of resistance to infection in mice and rats. The reactivity of candidate antigens at the cellular level with human T and B cells will be assessed using samples from newly collected endemic cohorts to discover protective cellular responses. The combined data will point out the best candidates for an effective prophylactic vaccine against schistosome infections.

The specific scientific and technical objectives are:
- Analysis of stage-specific gene expression profiles of S. mansoni life stages
- Bioinformatics-based selection of skin-stage schistosomula proteins with vaccine candidate potential
- Generation of glycomic profiles of S. mansoni life stages
- Creation of natural Schistosoma glycan micro arrays to enable serum screening
- Immunological screening of the post-genomically selected protein and glycan antigens by HT technologies
- Establishment of an in vitro killing and opsonisation assay of schistosomula
- Selection of antigens with high correlates to protection by in vitro killing potential
- Selection of antigens with high correlates to protection by in vivo rat model
- Engineering of trifunctional antibodies and testing in vitro and in vivo mouse model
- Establishment of new cohorts from endemic areas
- Selection of antigens with high correlates to protection and vaccine potential by testing in vitro T- and B-cell responses in human cohorts
- Dissemination of results in peer reviewed international journals
- Creation of a nucleus of African scientists active in the field of innovative vaccine research


Project Results:
INITIAL VACCINE TARGETS

TheSchistoVac has selected a large set of potential targets for an effective immune response against schistosome larvae and/or adult worm stages. These initial targets have been selected on the basis of bioinformatics analysis of gene expression profiles that predict which proteins are expressed in the schistosome life stages most vulnerable to immune attack, and by the mass spectrometric analysis of the glycan antigens expressed in these same life stages. These protein and glycan targets were produced by recombinant expression and chemical synthesis, respectively, for functional and immunological testing in in vitro and in vivo.

Genes and proteins:
Based on the transcriptome analysis of the schistosome life stages, 46 putative vaccine candidate proteins with the desired expression profiles and surface expression or secretion characteristics have been identified and prioritised. Twenty seven of these genes have been cloned, and 15 recombinant candidate proteins have been expressed for direct use in serum antibody screenings, rat immunisations and the assessment of cellular response characteristics of B- and T-cells.
The bioinformatic analysis of all S. mansoni gene products that are potentially secreted/excreted or expressed on the surface of the parasite produced three datasets originating from the microarray analysis of the parasite lifecycle:
- Dataset 1: cercarial/schistosomula-enriched transcripts (as defined by statistical analysis)
- Dataset 2: platyhelminth-specific transcripts expressed only in the vertebrate host lifestages (as defined by Network analysis)
- Dataset 3: transcripts lacking human orthologs expressed only in the vertebrate host lifestages (as defined by Network analysis)
We employed three bioinformatic filters to identify from these datasets all the gene products potentially secreted/excreted or expressed on the surface of the parasite:
- Genomic mapping
- Secretion signal/transmembrane region identification
- Protein sequence examination
The final bioinformatic filters aimed to identify and exclude proteins with the following criteria: difficult to recombinantly express, with similarities to allergens, with incomplete gene predictions, already represented in the list or unlikely to be present on the surface of the parasite by domain annotation. Through this selection, 39 S. mansoni in initial vaccine candidates were found. Analysis of the classes of proteins selected shows a large number of the vaccine candidates (12 candidates) are from “unknown protein families”. Further comparison finds that none of these twelve proteins are related to each other, so represent twelve uncharacterised protein classes. Of the vaccine candidates from characterised protein families, four families are represented more than once: tetraspanins (3 vaccine candidates), CD59-like proteins (3 vaccine candidates), saposins (3 vaccine candidates) and MEG proteins (6 candidates). To facilitate the production of recombinant proteins, the following genes were cloned: SmKK7, SmCSS-1, SmCD59a, SmTSP7, SmTSP6, SmCD59b, Smp_075420, SmCD59c, Smp_194860, SmMEG8a, Smp_150330, ShTSP6, ShCD59c, ShMEG8a, Smp_121930, Smp_166340, Smp_093790.
For eight proteins Standard Operation Procedures (SOP) have been established for protein expression and purification (SmKK7, SmCSS-1, SmCD59a, SmCD59b, SmCD59c, SmTSP6, SmTSP7 and smp_075420) and they were produced as recombinant antigens at the mg scale to allow application in the in vitro and vivo models for schistosome vaccine testing. Average yields of purified protein per litre starting culture range from ~500g pure soluble protein per litre starting culture (SmKK7) to ~9mg pure resolubilised protein per litre starting culture (SmTSP7).

Glycans and glycans arrays:
Schistosome glycans are highly antigenic, strong antibody responses are observed in schistosome infections against glycans and therefore they form potential vaccine targets. TheSchistoVac has determined the developmental glycan expression profiles of S. mansoni by mass spectrometric approaches, and identified the structures of N-glycans, O-glycans and glycolipid glycans expressed by cercariae, developing schistosomula and adult worms. A gradual change in glycosylation, in particular at the surface of the schistosome was observed during development of schistosomula, but with (multi-)fucosylated glycan motifs constantly being present at the surface of the parasite. Hence those type of glycans constitute potential targets of an immune reaction to the primary target of TheSchistoVac, the skin stage schistosomulum.
The hundreds of glycans isolated during these identification studies were used to construct glycan-microarrays suitable for screening with sera to simultaneously measure antibody responses against the whole range of printed glycan antigens. The largest array we constructed of fractionated N-glycans from cercarial, adult and egg stages contained isolated schistosome glycan fractions and additional synthetic and natural glycoconjugates resulting in a total number of 1143 samples that were printed in triplicate on the glass slides. A protocol was developed allowing to use the array for detecting antibodies to the different antigenic glycan fractions in serum, yielding a SOP including washing, blocking and incubation conditions, fluorescence scanning settings, and quantification and analysis parameters. Hierarchical clustering analysis and principal component analysis were found suitable for comparisons of responses in different groups of sera.

ANTIBODIES IN SERA OF SCHISTOSOME-INFECTED COHORTS

Anti-protein antibodies:
High throughput (HT) screenings have been performed of all recombinant proteins produced using sera from a study cohort in Uganda that includes populations that are putatively susceptible or resistant to S. mansoni re‐infection after treatment. Several of the recombinant proteins indicated the desired antibody reactivity characteristics: robust IgG response while no IgE reactivity, or no reactivity at all (but possibly inducible by vaccination).
rSmCSS1 - Infected individuals produced high levels of IgG1 (IgG4 & IgE responses were rare) The IgG1 levels did not change after praziquantel (PZQ) and were not correlated with re-infection levels 8m or 2y after PZQ. IgG1 also detected in uninfected Europeans.
rSmKK7 - >70% of the infected cohort produced IgG1 to rSmpKK7 (IgG4 & IgE responses were rare). IgG1 levels did not change after PZQ and were not correlated with re-infection levels.
Smp_075420 - The antibody response to this antigen was predominantly IgG1 and a strong IgE response was absent. There were no associations between antibody levels and re-infection either at 8 months or 2 years post PZQ. All antibody levels decreased following praziquantel (PZQ) treatment. Antibody levels were not correlated with age or pre-PZQ intensity of infection.
SmCD59b/Ly6b - 25% of the infected study cohort produced an IgG1 response to SmCD59b . IgG4 and IgE responses were not seen. There were no associations between IgG1 levels and re-infection either at 8 months or 2 years post PZQ. IgG1 levels decreased following treatment and the response was not associated with age or pre-PZQ intensity of infection.
rSmCD59a/Ly6a, rSmTSP6, rSmTSP7 – In each case, extremely low levels of specific IgG1, IgG4 and IgE detected in the majority of infected individuals. No change with PZQ treatment or re-infection.

Taken together, IgG1, the Ab isotype of interest, was measurable above assay detection limits for SmKK7, SmLy6b/CD59b and Sm075420. When analysed in respect to a cut-off derived from a panel of uninfected sera, 77%, 24.5% and 36.5% respectively, of individuals from the endemic cohort were classed as responders to these antigens. The levels of IgG1 to all three antigens dropped after treatment, significantly for SmLy6b and Sm075420, indicating that live infection supports the production of IgG1 to these antigens. None of the IgG1 responses were associated with natural immunity.
For all antigens there were weak relationships between infection intensity and having a measurable IgG4 response. It was not the same individuals who raised an IgG4 response to all antigens however, it appears inherent within endemic populations that a few individuals will raise this response to schistosome antigens, and this association should not preclude proteins from inclusion in vaccination experiments.
For all antigens, there were very few individuals who responded with IgE, and for the majority of the antigens responding with IgE was not associated with age or infection intensity. However, there were weak but significant associations between responding to IgE and age for TSP7 and Sm075420.
SmKK7, appears a promising vaccine candidate: inducing a schistosome-specific IgG1 response, with little evidence for IgE responsiveness. However, despite slightly lower IgG1 response rSmCD59a/Ly6a, rSmTSP6, rSmTSP7, SmCD59b/Ly6b are all considered potential vaccine candidates in view of the virtual absence of potentially dangerous IgE responses and their bioinformatics characteristics.

Anti-glycan antibodies:
The glycan microarrays have been screened first with a preliminary small serum cohort to assess the quality of the arrays and fine‐tune the protocols for incubation, data analysis and glycan fraction printing. On the basis of this, an improved 2nd generation glycan array has been established which was subsequently screened with a much larger Ugandan cohort of 352 sera from 176 infected individuals (2 samples each, baseline and 9 weeks post-treatment) taken from the cohort used in the protein HT screening. This allowed more intricate analysis of the IgG and IgM responses to specific glycan motifs related to multiple parameters including resistance to reinfection. Endemic groups can be discriminated on the basis of their antibody response to sets of antigenic glycans isolated from the different parasite life cycle stages, with specific response clusters forming for sera as well as for glycans. We have observed differences in IgG as well as IgM responses with IgG to glycans being significantly higher in older individuals compared to the age groups that are most susceptible to reinfection while IgM is lower. Especially IgG to IgM ratio show pronounced differences between the different age groups. Major differences were observed for cercarial lipid glycans as well as egg N-glycans. These glycans contain the antigenic fucosylated glycan motifs that have been tested in the rat vaccination model.
In addition, to yet further improve the array for future use we have isolated and fractionated another class of glycans (O-glycans) through a modified hydrazinolysis method allowing reaction of the released O-glycans with a linker suitable for array construction. Using the resulting Schistosoma O-glycan microarray we found that that O- glycans is highly antigenic in schistomiasis. Significant antibody responses were observed both in human natural infection sera as well as in experimental animal vaccination sera that were tested in parallel to TheSchistoVac research. O-glycans arrays should be used for further profiling of human serum or plasma cohorts to identify antigenic Schistosoma O-glycans and possibly, O-glycan vaccine candidates. We concluded that O-glycans should be taken into consideration as vaccine candidates.

IN VITRO AND IN VIVO PROTECTION MODELS

In vitro binding and opsonisation of schistosomula:
In vitro schistosomula antibody adherence and antibody-mediated cellular killing assays have been set up and were used to test the adherence of serum antibodies from immunised rats to confirm the potential of antibodies to recombinant proteins to act on skin stage schistosomula. Antisera against the selected recombinant protein antigens where in general not or only weakly binding to the surface of schistosomula. A number of candidate antigens however, including SmKK7 and SmCD59a/Ly6a are present in the schistosomulum tegument according to proteomic studies. To help revealing hidden surface antigens and to enhance the efficiency of the protein-specific antisera in the in vitro killing assay, the drug-assisted in vitro approach was established and utilised to show that antigens do become accessible to antibodies after drug treatment. We found that drug treatment of schistosomula in vitro with praziquantel, artemether or oxamniquine leads to exposure of CD59a at the surface and – importantly - increased binding of rat-infection sera, thereby making otherwise hidden antigens accessible to the immune system and increasing their vaccine potential. Similarly, a panel of anti‐glycan mouse monoclonal antibodies (mAbs) generated from Schistosoma-infected mice was screened for binding to schistosomula in vitro to confirm the presence of antibody-accessible glycans at their surface. Most mAbs that recognise fucosylated variants of HexNAc residues, also bind to the surface of schistosomula at least up to 3 days old, supporting the hypothesis that these type of glycans can form a target for antibody-mediated immune attack of schistosomula. Two anti-glycan mAbs that recognise multifucosylated glycans, 114-5B1 and 128-1E7, in particular appeared to bind and damage schistosomulum tegument in the in vitro assay. There is now an urgent need for positive controls in the rat model to enable definitive conclusions to be drawn.

Rat vaccination/protection model:
A rat model of schistosome infection has been set up. In the rat model, which is particularly suitable to study immunity to early schistosomula stages (important for a prophylactic vaccine) the recombinant proteins positively selected on the basis of the serum screenings have been used to A) generate rat immune sera, and B) test the effect of immunisation on worm burden after a challenge infection with S. mansoni.
Concerning the IgE response of infected rats, we only detected significant IgE against SmKK7 in infected Brown Norway (BN) rats. When the IgG responses were explored, we detected significant IgG antibodies against SmKK7, SmCSS1 and SmCD59a/Ly6a both in infected Fischer and BN rats with higher levels in infected BN rats. A very weak IgG response was detectable against TSP6 and TSP7 in BN rats. Beyond the fact that recombinant proteins are recognised by sera from infected rats, these results confirmed the correct folding of the expressed proteins.
The rat model was used as our main in vivo model for testing the protective effects of immunisation with our vaccine candidates on challenge infection with schistosomes. The vaccine candidates tested in the immunisation/challenge model were SmKK7, SmCD59a, SmCD59b, SmTSP6, SmTSP7 and Sm_07450. Immunisations were performed with Th2 adjuvant (Alumn) and in some cases in addition with Th1 adjuvant (CFA). None of the recombinant vaccine candidates provided significant infection against a challenge infection when used in immunisations together with Alumn as adjuvant. SmKK7, SmCD59a and SmCD59b were in addition tested with CFA adjuvant (boosted with repeated injections in FA), and SmKK7 gave initial promising results. The protective effect was however not reproducible in 3 independent experiments (exp1 – 34% worm burden reduction p < 0.05 exp 2 – 35% w.b. red. but not significant, exp 3 – no difference). In addition to the recombinant proteins two synthetic glycan-albumin conjugate antigens were tested in the rat model, each with CFA adjuvant.
Finally, as planned, we performed protection experiments with glycoconjugates recognized by human sera.

ESTABLISHMENT OF COHORTS AND CELLULAR IMMUNE RESPONSES TO VACCINE CANDIDATES

General:
Through capacity building of scientists from African partner institutions, TheSchistoVac has generated maps of the study areas in 4 schistosome endemic areas and provides full data on the prevalence of S. mansoni and S. haematobium infections in age graded manner. Moreover, through the project it has been possible to understand the local sociocultural sensitivities and beliefs that would need to be tackled for sensitising the communities for future vaccine interventions and/or new treatments. This information is one of the important products of TheSchistoVac in Gabon, Ghana, Kenya and Uganda. For example, in Kenya it was surprising to realise that an area where much of the year faces draughts with little water in the rivers, there is still considerable transmission of S. mansoni. Such information through TheSchistoVac project has reached the national ministries of health.
The field activities have resulted in detailed maps of the studied endemic areas where infection prevalence is provided in a two dimensional manner and can be used for future studies and control measures. In addition through TheSchcistoVac a number of Standard Operating Procedures (SOPs) have been generated: data collection using Geographic Information System (GIS); Collecting socio-demographic data; Study information and consent forms, Sample collection and storage; Detailed laboratory procedures for collection of blood, isolation of cells and immunological studies. The laboratory procedures need to be present in endemic countries and in the appropriate sites that would be ready to start conducting further immunological studies and trials in the future. Preparing sites to bring the end products of programmes such as TheSchistoVac are crucial for the ultimate vaccine development. Phase I trials, usually conducted in Europe or the USA, need to then be taken to the endemic regions where the target population resides. The roadblocks to such activities can be the lack of preparedness of the local institutions. Therefore, one of the important achievements of TheSchistoVac was the start to prepare the SOPs that would allow the study of cellular immune responses:
- Culture for characterization of T cells by flow cytometry
- Measurements of cytokines in cell supernatants by multiplex and by ELISA
- ELISPOT assay
- Characterization of B cell memory subsets
- Characterization of exhausted B cells
- Preparation of schistosome antigen extracts suitable for cellular assays
- Antigen specific intracellular cytokine staining (ICS) to indicate cytokine production by CD4+ T cells

The most important SOP that was developed within TheSchistoVac project was for the identification of antigen reactive T cells. This is considered as one of the most challenging steps that needs considerable work to optimise as the frequency of cells reactive to single antigens are generally very low. For the field of schistosome research this has now been achieved with TheSchistoVac and might apply to other areas of helminth research. To optimise the ICS protocol for detection of antigen-specific Th1 and Th2 cytokines. Several variables, such as the total length of culturing, the ideal time point for the addition of protein export , resting and co-stimulation, have been tested with various antigen-specific stimuli on various donors from countries were helminth infections are endemic or not. The difficulty of detecting some of the cytokines has been resolved by using multiple antibodies with the same fluorescent label to intensify signal. For example for Th2 responses antibodies to all three cytokines, IL-4, IL-5 and IL-13 were added to the cocktail but all three were labelled with a similar fluorochrome.
Using all generated SOPs, several lines of activities were undertaken to first understand immune responses associated with schistosome infection by comparing infected with uninfected and to assess the responses to novel vaccine antigens identified by TheSchistoVac in the so called “resistant” and “susceptible” subjects which were identified through an algorithm developed by University of Cambridge based on the IgE responses to adult worm antigens.

T cell responses associated with schistosome infection:
The protocols to characterize effector and central memory T cells based on the expression of CD3, CD4, CD45RA, CD27 and CCR7 which were established during TheSchistoVac and applied to endemic sites. The analysis of different memory T cell subsets, i.e. effector and memory T comparing European and individuals from rural and urban areas of endemic countries, showed that the CD4+ T cell memory pool is enlarged in more rural areas, at the expense of the naïve compartment. When analysing within a rural area the difference between schistosome-infected and -uninfected subjects, the naïve T cell compartment was found to be significantly lower in the infected subjects. Data from comparing immune responses before and after removal of schistosomes by praziquantel which confirm that infection and not other confounding elements contribute to the immune profile seen, indicate that effector memory T cells are lower before infection is removed. Interestingly, regulatory T cell responses which were significantly higher in schistosome infected subjects compared to uninfected, decreases after removal of praziquantel treatment suggesting that higher regulatory T cell responses are associated with effector memory responses to schistosome antigens. These data are important in the context of understanding the immune response development in endemic areas where target population is being tested for their reactivity to vaccine candidates. In practical terms, these data indicate that it might be important to treat and remove schistosome infections prior to vaccination. Moreover, the data also provide argument for the testing of vaccine candidates in those exposed but uninfected.

Cytokine responses to schistosome antigens:
Upon availability of the vaccine candidates following testing for endotoxin contamination and purity, T cell reactivity in the four endemic regions was studied. The study area in Ghana has had the problem that in between the survey and actual recruitment, mass treatment was offered to the community and although we have recorded history of praziquantel intake, this has meant that we have had a lot of difficulty to identify the putatively resistant and susceptible subjects.

Consideration of general responses:
As positive controls the whole schistosome adults worm or egg antigen extracts were used for Th2 response calibration while mycobacterial purified protein derivative (PPD) was used for Th1. The responses to PPD were indeed dominated by Th1 responses in all sites. The cytokine responses to total adult worm antigens and to soluble egg antigens was dominated by Th2 in all sites. The responses to the recombinants was detectable and varied but much lower Th2 responses were seen. Some individuals showed a mixed response to SmKK7 and SmLy6A/CD59a , however, it should be noted that the Th1 to Th2 propensity was much in favour of Th1. Others responded to SmLy6B/CD59b and SmTSP7 by IFNϒ production in general. These results are preliminary and would need to be confirmed in extensive data analysis were not only median responses but also responder, non-responders are compared.

Responses in the study population categorized as resistant and susceptible:
Several ways can be used to identify resistant and susceptible subjects in schistosome endemic areas. In one of the partner institutions, years of experience considering subjects who are resistant to reinfection and taking into account their water contact history, has shown that IgE responses to adult worm antigen and in particular SmTAL-1 can be strong predictor of resistance to reinfection. In South America, another group of scientists have identified resistant subjects as those who despite a life-long exposure to infected waters do not become infected with schistosomes. These subjects show immune responses to schiostosomes indicating indeed that there is exposure to these parasites, moreover, their water contact has been recorded and again shown that they indeed exposed to infested water bodies. Using the categorization of the resistant and susceptibles using IgE to adult worm antigens, the following number of subjects were identified :
- In Gabon 16 susceptible and 12 resistant subjects were studied
- In Ghana a total of 53 individuals were analysed but due so far of these 2 subjects were categorized as susceptible and 18 as resistant.
- In Kenya 24susceptible and 31 resistant subjects were analysed.
- In Uganda 50 susceptible and 50 resistant subjects were studied.

Due to the numbers in Ghana (which were partly due to the problem of mass treatment in the community between surveys carried out and baseline measurements) the results for Ghana can not be used currently and more work needs to be done on larger number of samples to identify more susceptible individuals.

The responses to the candidate antigens were as follows: The preliminary results so far show that responses to SmKK7, SmLy6a/CD59a and SmTSP6 currently show no strong consistency between the countries. It should be noted that for all analyses, the data need to be scrutinized in terms of the quality of flow cytometry, gating and background noise which although are so far considered adequate have to go through formal assessment.
In Gabon and Uganda, the pro inflammatory cytokines IFNϒ and TNF as well as the pro-proliferative cytokine, IL2 was higher to the same two antigens, namely SmLy6B/CD59 and SmTSP7 in the resistant subjects compared to the susceptibles. This is the first time ever that immune responses studied across different countries are showing consistent cellular immune responses where not the Th2 but Th1 responses are more predominant in those who are predicted to be resistant to infection. In Kenya, such responses could not be confirmed, however, it was noted that in susceptible subjects the response to SmTSP7 was dominated by Th2 which might be in line with what has been seen in Gabon and Uganda ie Th1 to Th2 balance is in favour of Th1 in resistant subjects. We consider these results extremely promising and suggest future studies to expand this not only to larger numbers of “resistant” and “susceptibles” but to also test these in other areas where the so called naturally resistant subjects are studied such as in Brazil.


B cell responses associated with schistosome infection:
Another major progress is in the area of development of methods to characterize B cells during chronic helminth infection. Work within TheSchistoVac has allowed the development and application of novel protocols for studying B cells using CD27+ and ‘exhausted, atypical’ CD27- memory B cell (MBC) subsets in Gabonese schistosome-infected versus uninfected individuals. In addition the regulatory Breg subsets (CDd1hi, CD24hiCD38hi and CD24CD27) have also been studied in the context of schistosome infection. These protocols have been applied to study the effect of being infected with schistosomes on B cells and are now ready to be applied to individuals who are considered as resistant or susceptible to schistosome infection
The findings of the project regarding the effect of schistosome on B cells are that IgG+ (but not IgM+ or IgA+) B cells were increased, which was mostly accounted for by the ‘exhausted’ (or atypical or double negative - DN) CD27- MBC subset in infected individuals. At the functional level, isolated B cells from schistosome-infected subjects showed higher expression of the activation marker CD23 upon stimulation, but lower proliferation and TNF-α production in response to TLR-9 ligand with or without BCR stimulation. Increased levels of IL-10 producing CD1dhi B cells in infected compared to uninfected individuals were measured. Interestingly, co-cultures of B cells with CD4+ T cells showed increased Treg cells levels and IL-10-producing T cells in the case of B cells from helminth-infected donors, while IL-17 and IFNg production was reduced. Importantly, CD1d+ B cell depletion ablated IL-10 production in the T cells, but not the FoxP3 Treg cells, suggesting that there was more than one active Breg subset in schistosomiasis.
When considering antibody production, schistosome-infected children were found to carry higher frequencies of IgG+ double negative (CD27−IgD−) MBCs compared to uninfected children, whereas no differences are observed in the levels of IgM+ or IgA+ DN MBCs. Similarly, no differences were observed in total serum IgM, IgA nor IgG1, IgG2 and IgG3, whereas significant differences were only observed in IgG4. Interestingly, serum levels of IgG4 were significantly decreased following treatment and correlated with a concomitant decrease in the frequency of the DN MBCs, suggesting that the increase in IgG+ DN MBCs may be predominantly due to an increase in IgG4-expressing B cells. As IgG4 is associated with susceptibility, these protocols can now be applied to the characterization of the cell subsets in so called “resistant” and “susceptible” subjects. Taken together, evidence was generated on the increased frequencies of regulatory B cells, differences in the exhausted B cells and increased IgG4 producing B cells being associated with schistosome infection and these parameters would need to be considered within the context of resistance during natural infection or during vaccination.

Foreground and Future to the cellular studies both T cells and B cells:
So far the activities within TheSchistoVac have provided the results/foregrounds:
- Detailed maps of the endemic study areas
- SOPs for use in endemic areas and the laboratories associated with TheSchistoVac
- Specific protocol for the detection of antigen specific T cell responses in schistosomiasis
- Data on how schistosome infection affects T and B cell responses
- Data on the responses to TheSchistoVac novel vaccine candidates: For the first time data on higher Th1 & pro inflammatory/Th2 responses to a single antigen in 3 endemic region in putatively resistant subjects.

These data in the context of data generated at the different stages of TheSchistoVac in different workpackages will be discussed next. The identification of new antigens as vaccine candidates based on bioinformatics approach, the success in expressing them in sufficient quantities and in a pure and endotoxin free manner, is a major achievement of the project. The production of the antigens was based on information obtained on safety, i.e. the IgE reactive antigens were disqualified. The promising antigens so far SmLY6B/CD59b and SmP7 when considering the cellular immune responses, did not show any protection in animal models (rat or mouse) and did not show superior IgG1 reactivity in so called “resistant”subjects. There are a number of issues to consider when moving to the next stage using the findings of TheSchistoVac. One is the importance of including a positive control when conducting the animal studies. By this it would mean taking either antigens that so far have been reported to give higher than 60% protection, such as SmTSP2 and Sm29. An alternative would be to use irradiated cercariae which gives very high levels of protection. The benchmarking is essential to be able to establish with certainty whether the antigens that are promising with respect to cellular immune responses show any protective activity against positive controls. In addition the studies using baboon would also be an important step forward. Next, the reactivity in so called “resistant” and “susceptible” subjects would need to consider the fact that these subjects were selected based on IgE to adult worm antigen. Additional criteria need to be developed for the so called resistant and susceptibles which does not depend on IgE. Moreover, it would be important to test these antigens against the naturally resistant subjects from Brazil.
Finally, given the contribution of regulatory T and B cell responses to the overall immune response in infected subjects, it would be important to use samples from treated subjects in order to circumvent the regulatory responses.

Potential Impact:
IMPACT, DISSEMINATION AND EXPLOITATION OF THE RESULTS OF THESCHISTOVAC

The need for a vaccine:
Although schistosomiasis is treatable, the current treatment of choice does not provide an optimal strategy for controlling or eliminating the disease. Praziquantel (PZQ) the treatment of choice has been used in annual treatment of school-aged children but the results of more than a decade of using this approach has not produced satisfactory results in terms of full control or elimination of schistosomiasis. A survey of over 400 experts in the field indicates that most feel that schistosomiasis will never be eliminated using only mass drug administration with PZQ because of the problem of post-treatment re-infection, requiring frequent treatment rounds. Moreover, PZQ is mainly effective against the adult worms. Therefore, adequate schistosomiasis control would require the repeated administration of hundreds of millions of drug treatments on a regular basis. Then we need to consider the effect of mass treatments which may eventually induce PZQ-resistant strains of schistosomes. Although new drugs would be useful in case resistance develops, even new drugs cannot prevent rapid re-infection after treatment.
Additional factors are playing an important role in understanding the schistosome transmission. Infection occurs as a result of contact with water, and without access to piped water or any other form of potable water, the schistosome endemic communities remain dependent on their water bodies and therefore there is continual schistosome exposure. As part of TheSchistoVac project numerous community meetings have been held to improve community awareness on this issue. However, it also needs to be taken into account that many in endemic regions are fishing communities, their contact with water bodies is inevitable. The value of the water bodies can be deeply engrained in the community. Not only is the water body a source of water for consumption but also for basic everyday activities of individuals in communities such as bathing, washing and an avenue for transportation. In addition it is important to note that the importance and reverence of the water bodies in the community means that even if an alternative water source would be established to provide potable water, the deep connection between the river and the community would hamper public health interventions aimed at eliminating exposure to the river. Therefore, the situation is more complex and reinforces the importance of an effective vaccine.
In addition one needs to consider that schistosomes ensure their long term survival by a sophisticated, almost perfect, array of immune evasion mechanisms. However, despite their strategies for survival, careful human studies in disease endemic areas over the last 30 years have shown that an important form of natural human immunity can occur against schistosomes and importantly the irradiated cercaria can lead to strong protective immunity in animal models, including primates, vouching for the feasibility of vaccine development.
A number of groups worldwide are active in development of a vaccine against schistosomiasis. Reported promising antigens in recent years are SmTSP2, Smp80 and Sm29 which have shown protective results in animal models that are above 40%. However, with the exception of TSP2, which in a small study was tested against a number of human sera, none have been analyzed in any detail, using human sera or cells to establish the immunological recognition in humans. One of the important issues that is now emerging is the importance of building a pipeline of vaccine candidates which can be put together as multivalent vaccine to induce strong immunity to a complex, multicellular organism such as schistosomes. With this in mind, TheSchistoVac project has been timely and important in identifying a series of novel antigens that can enter the pipeline. Moreover, TheSchistoVac has combined several disciplines and has expressed as well as tested the vaccine candidates in several testing platforms in parallel.

Societal impact:
With respect to the preparation of the field activities, it has to be noted that TheSchistoVac has had important impact at the community level and the target population and at the level of local institutions in terms of capacity building. The communities involved have been consulted extensively, and community workers have been trained to participate in surveys and in the provision of treatment for schistosomiasis. Moreover, there has been consultation with the heads of the communities regarding the importance of exposure to the water bodies as source of infection. During the visits it has also become clear that control of schistosomiasis can be more complex when it comes to avoiding the source of infection, a point that would need further attention by ministry of health which has been informed by reports prepared by the research teams. In addition it was noted that efforts would need to be put into preparedness in terms of introducing a new vaccine into the community. This means that a strong basis would need to be available for community consultation and for preparation and implementation of future schistosomiasis vaccine trials in the communities.
The presence of the research team which consisted of medical and para medical staff, translated into attending to any medical problems that participants reported. If unable to help, the patients were referred to the local health centers. The results of the schistosome infection diagnoses were reported back to all tested subjects and treatment provided. The question whether this has any concrete impact in terms of protecting the subject from becoming re-infected is doubtful as outlined above but the medical and para medical team acted in accordance with the national guidelines.
The contribution and impact of TheSchistoVac on the local institutions can be summarized as capacity building and generation of teams that are equipped to work on several different health related topics that share methods such as geo-surveying, parasitology diagnosis, research methodology such use of statistics, humoral immunological and molecular biological methods and finally the cellular immune responses. The awareness that vaccines are the most effective way forward for a number of poverty related and neglected diseases would mean that future trials are expected to be conducted in Africa and a well-equipped and well-trained team will be essential. TheSchistoVac project has been instrumental to the training of 3 technical staff, two MSc students and 5 PhD students from endemic regions. The two MSc students have recently entered a PhD programme. TheSchistoVac has also contributed to the establishment of South to South collaborations where expertise such as malacology has been exchanged as well as cellular immune measurements.

Scientific impact:
Identification of stage-specific schistosome antigens and safety regarding IgE reactivity:
Taking the bioinformatics approach based on sound scientific reasoning, the project has identified 27 antigens of interest which were expressed in early schistosomula, could be present on adult worms but importantly not on eggs. Of these 15 could be expressed and due to the timeline of the project, those expressed in sufficient quantities were further examined with respect to reactivity with IgE antibodies. The consortium could identify 5 promising antigens which were produced in sufficient quantities for animal testing as well as studies of human immune responses. These antigens SmKK7, SmLy6a, SmLy6b, SmTSP6 and SmTSP7 were novel antigens. In parallel, the field sites were identified and accurate maps presenting the data on infection two dimensionally were prepared. In conjunction, the training of scientists from African centers as well as European scientists regarding all technologies required for the completion of the goals of TheSchistoVac were completed.
The impact of the project in terms of identification of the 27 possible antigens, is scientific. This would form a pipeline where possible candidates would need to enter different phases of vaccine development. The next step achieved in the project where 5 identified genes were cloned and expressed successfully in larger quantities, enters the schistosome vaccine pipeline where antigens can be tested in different field sites, different animal models and in collaboration with other vaccine antigens could form parts of a subunit vaccine.
An interesting aspect of TheSchistoVac antigen discovery was the testing of carbohydrate structures that are present on schistosomes as possible vaccine candidates. The glycoconjugate carrying Glycan 1 (CODED) was identified as immunogenic and unlikely to react with IgE and induced a protective response in the rat model.

Novel antigens tested in vitro and in vivo -
The in vitro killing assays have been set up during TheSchistoVac to examine whether promising antigens could be identified where humoral responses would lead to effective killing of schistosomula. The results have shown that when combined with drugs that can damage the parasite tegument, antibodies which show no direct reactivity to tegument, can bind the surface of the parasites. This has currently no direct measureable impact.

The testing of the vaccine candidates in rats and mice to assess level of protection that can be achieved in vivo, has only shown some promise for SmKK7 which respect to the selected protein antigens. One of the major issues regarding this is the availability of a positive control. In this regard other antigens such as TSP2 which are reported to show more than 60% protection or irradiated cercaria could be used as positive control to allow the assessment of the level of protection that our novel antigens induce.

The endemic regions -
The availability of detailed maps of the endemic study areas with information on where the infection loci are, can be used by local health authorities and ministry of health which are in charge of the annual praziquantel treatment and therefore can direct efforts at the community.
The SOPs generated, some unique, during TheSchistoVac for use in endemic areas and the laboratories associated with the local institutions will be used in future studies that would require similar methods of analyses. For example, studies looking for hookworm vaccines, or studies that involve phase I or II trials of existing schistosome vaccines can use a number of SOPs that have been developed during the project saving time, energy and money. TheSchistoVac helped the local research capacity to be updated and to allow the groups to get involved in multicentre studies. It also equips them with the technological information that would be needed if they were to participate in vaccine trails.

Immune recognition of the vaccine candidates in humans -
TheSchistoVac has generated unique data showing higher ratio of Th1 & pro inflammatory / Th2 responses to a single antigen in 3 endemic region in putatively resistant subjects. These antigens are SMLY6B/CD59b and SmTSP7. However, when tested with sera from resistant and susceptible subjects, there was no indication that resistant subjects produced higher levels of IgG antibodies. Both of these results, cellular and humoral, would need to be further confirmed in additional cohorts and defined as resistant and susceptible based on different criteria. However, it is possible that different antigens are capable of inducing a strong antibody response and CD4+ T cell responses. The latter would be important criteria for activation of cells such as macrophages to induce immune attack while the former for the opsonisation.

These data have scientific impact where the research community working on the development of schistosome vaccine can benefit from. This could benefit the planning of a subunit vaccine where a number of antigens with different effect are combined:
-antigens that induce protective IgG isotypes
-antigens that induce a strong T cell response with Th1 and pro inflammatory signature

Summary of the scientific impact of the project -
The identification of new antigens as vaccine candidates based on bioinformatics approach, the success in expressing them in sufficient quantities, pure and endotoxin free, are major achievements of the project. The production of the antigens was based on information obtained on safety, meaning the IgE reactive antigens were disqualified. The promising antigens so far, SmLy6B/CD59 and SmTSP7, when considering the cellular immune responses, did not show any protection in animal models (rat or mouse) and did not show superior IgG1 reactivity in so called “resistant” subjects. There are a number of issues to consider when moving to the next stage using the findings of TheSchistoVac. One is the importance of including a positive control when conducting the animal studies. By this it would mean taking either antigens that so far have been reported to give higher than 60% protection, such as SmTSP2 and Sm29. An alternative would be to use irradiated cercariae which gives protection levels exceeding 80%. The benchmarking is essential to be able to establish with certainty whether the antigens that are promising with respect to cellular immune responses show any protective activity. In addition the studies using baboons would also be an important step forward. Next, the reactivity in so called “resistant” and “susceptible” subjects would need to consider the fact that these subjects were selected based on IgE to adult worm antigen. Additional criteria need to be developed for the so called resistant and susceptibles which does not depend on IgE. Moreover, it would be important to test these antigens against the naturally resistant subjects from Brazil. In addition, with the idea of generating subunit vaccines, it might be important to combine antigens with different effects. The future might need a combination of antigens that induce strong and protective IgG responses with antigens that are particularly strong in inducing a Th1 or pro inflammatory responses that are able to activate macrophages to kill the parasite with opsonized antibodies.
Finally, given the contribution of regulatory T and B cell responses to the overall immune response in infected subjects, it would be important to use samples from treated subjects in order to circumvent the regulatory immune responses which decline once infection is removed.

These scientific results of TheSchistoVac have been disseminated through specific conferences and during meetings where researchers and sometimes industry were present.
The way forward will be to start preparation of a roadmap that will stipulate the following activities with the identified candidate vaccines:
- Set up collaboration with other consortia active in development of schistosome vaccine to allow testing in : different groups of resistant and susceptible subjects, in rat and mouse vaccination models using positive controls, other animal testing such as in baboons and macaque
- Discuss subunit vaccine design
- Prepare the target community by initiating sociocultural studies where vaccine acceptability is addressed
- Dialogue with the funding agencies such as the Dutch government, Gates foundation and EDCTP
- Partnership with an organization such as SABIN, which is a non-profit organization that promotes advances in vaccine development, delivery and distribution.


List of Websites:
The public website address of TheSchistoVac is www.theschistovac.eu

For further information about the project, its output, and its impact, contact the co-ordinators at the Leiden University Medical Center

Dr. Cornelis H Hokke - c.h.hokke@lumc.nl
Prof. Dr. Maria Yazdanbakhsh - m.yazdanbakhsh@lumc.nl

Department of Parasitology
Leiden University Medical Center
PO Box 9600
2300 RC Leiden
The Netherlands

phone +31 71 5265062